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Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation.

Srivastava P, Yang H, Ellis-Guardiola K, Lewis JC - Nat Commun (2015)

Bottom Line: The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water.This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction.Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

View Article: PubMed Central - PubMed

Affiliation: Coskata Inc. 4575 Weaver Parkway, Warrenville, IL 60555, USA.

ABSTRACT
Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor-acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

No MeSH data available.


Homology model21 of Pfu POP.The hydrolase domain is shown in green, the propeller domain is shown in grey and cofactor 1 linked at Z477 is shown in red. Sites of different mutations introduced into Pfu POP are shown as coloured spheres.
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f2: Homology model21 of Pfu POP.The hydrolase domain is shown in green, the propeller domain is shown in grey and cofactor 1 linked at Z477 is shown in red. Sites of different mutations introduced into Pfu POP are shown as coloured spheres.

Mentions: An extensive search of different protein X-ray structures in the protein data bank (PDB) led to the identification of several members of the prolyl oligopeptidase family as potential ArM scaffolds because of their roughly cylindrical shapes (30 × 60 Å) and large internal volumes (5–8 × 103 Å3) for cofactor enclosure19. This family includes POPs, dipeptidyl peptidases IV, oligopeptidases B and acylaminoacyl peptidases. All of these enzymes share a common fold comprising an α/β hydrolase domain, which contains a Ser-Asp-His triad for amide bond hydrolysis, capped by a β-barrel domain. We initially selected a POP from Pyrococcus furiosus (Pfu) as a scaffold for ArM formation because of its high thermal stability20. Despite the abundance of POP structures in the PDB, however, the structure of Pfu POP has not yet been solved; therefore, a previously reported homology model21 of this enzyme was used for initial engineering efforts (Fig. 2). An amber codon was introduced into the POP gene to replace the catalytically active serine (S477) with a Z residue (Z477), abolish the native proteolytic activity of the enzyme and position the cofactor centrally within the active site. A POP gene whose codon usage was optimized for expression in E. coli was used as a template for genetic manipulation, and the resulting scaffold, POP-Z, was expressed in high yield (>100 versus ∼10 mg l−g before codon optimization) with essentially quantitative Z incorporation. Unfortunately, however, no reaction occurred between POP-Z and 1. POP variants in which other active site residues had been replaced with Z proved similarly unreactive towards 1, but rapid reaction of surface-exposed Z residues was observed22.


Engineering a dirhodium artificial metalloenzyme for selective olefin cyclopropanation.

Srivastava P, Yang H, Ellis-Guardiola K, Lewis JC - Nat Commun (2015)

Homology model21 of Pfu POP.The hydrolase domain is shown in green, the propeller domain is shown in grey and cofactor 1 linked at Z477 is shown in red. Sites of different mutations introduced into Pfu POP are shown as coloured spheres.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525152&req=5

f2: Homology model21 of Pfu POP.The hydrolase domain is shown in green, the propeller domain is shown in grey and cofactor 1 linked at Z477 is shown in red. Sites of different mutations introduced into Pfu POP are shown as coloured spheres.
Mentions: An extensive search of different protein X-ray structures in the protein data bank (PDB) led to the identification of several members of the prolyl oligopeptidase family as potential ArM scaffolds because of their roughly cylindrical shapes (30 × 60 Å) and large internal volumes (5–8 × 103 Å3) for cofactor enclosure19. This family includes POPs, dipeptidyl peptidases IV, oligopeptidases B and acylaminoacyl peptidases. All of these enzymes share a common fold comprising an α/β hydrolase domain, which contains a Ser-Asp-His triad for amide bond hydrolysis, capped by a β-barrel domain. We initially selected a POP from Pyrococcus furiosus (Pfu) as a scaffold for ArM formation because of its high thermal stability20. Despite the abundance of POP structures in the PDB, however, the structure of Pfu POP has not yet been solved; therefore, a previously reported homology model21 of this enzyme was used for initial engineering efforts (Fig. 2). An amber codon was introduced into the POP gene to replace the catalytically active serine (S477) with a Z residue (Z477), abolish the native proteolytic activity of the enzyme and position the cofactor centrally within the active site. A POP gene whose codon usage was optimized for expression in E. coli was used as a template for genetic manipulation, and the resulting scaffold, POP-Z, was expressed in high yield (>100 versus ∼10 mg l−g before codon optimization) with essentially quantitative Z incorporation. Unfortunately, however, no reaction occurred between POP-Z and 1. POP variants in which other active site residues had been replaced with Z proved similarly unreactive towards 1, but rapid reaction of surface-exposed Z residues was observed22.

Bottom Line: The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water.This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction.Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

View Article: PubMed Central - PubMed

Affiliation: Coskata Inc. 4575 Weaver Parkway, Warrenville, IL 60555, USA.

ABSTRACT
Artificial metalloenzymes (ArMs) formed by incorporating synthetic metal catalysts into protein scaffolds have the potential to impart to chemical reactions selectivity that would be difficult to achieve using metal catalysts alone. In this work, we covalently link an alkyne-substituted dirhodium catalyst to a prolyl oligopeptidase containing a genetically encoded L-4-azidophenylalanine residue to create an ArM that catalyses olefin cyclopropanation. Scaffold mutagenesis is then used to improve the enantioselectivity of this reaction, and cyclopropanation of a range of styrenes and donor-acceptor carbene precursors were accepted. The ArM reduces the formation of byproducts, including those resulting from the reaction of dirhodium-carbene intermediates with water. This shows that an ArM can improve the substrate specificity of a catalyst and, for the first time, the water tolerance of a metal-catalysed reaction. Given the diversity of reactions catalysed by dirhodium complexes, we anticipate that dirhodium ArMs will provide many unique opportunities for selective catalysis.

No MeSH data available.